Effects of HLA-B27 on Gut Microbiota Gut microbiota are strongly implicated in the development of spondyloarthritis. Given the importance of HLA-B27 in disease predisposition, it has been hypothesized that it might select for one or a small number of arthritogenic gut microbes. We investigated this question and found, quite surprisingly, that the effect of this B allele is quite dependent on genetics and/or environmental influences. Genetically different rat strains expressing HLA-B27 and housed in different facilities develop very different patterns of gut microbial dysbiosis, despite remarkably similar immune-mediated inflammatory changes. More in-depth analysis of gene-gene, gene-microbe, and microbe-microbe interactions using a novel inter-omic approach has identified candidate organisms from different rat strains that may drive HLA-B27-mediated inflammation. Moreover, informatics analyses predict that the different microbiota may be causing similar metabolic effects, underscoring the function consequences of key microbiota rather than their taxonomy. Thus, genetic and environmental factors play a large role in determining effects of HLA-B27, consistent with an ecological model of dysbiosis rather than changes in one or a few organisms. Genetic Contributions to Early Onset Axial Enthesitis-Related Arthritis To test the hypothesis that there are rare variants or de novo gene mutations that lower the age of onset of axial spondyloarthritis, we have been performing whole exome sequencing of affected children and their unaffected parents. Several candidate mutations have been identified, and the project is continuing. This follows on our previous report of a de novo germline gain-of-function MYD88 mutation in a child with severe destructive polyarticular juvenile arthritis. The mutation leads to gain-of-function activity in this adaptor protein for TLR and IL-1 receptor signaling, and increases NF-kB activation. This germline MyD88 mutation affects hematopoietic as well as non-hematopoietic cells (fibroblasts) and is likely to contribute to the development of arthritis, although effects on the immune system are expected to be complex. The Interaction Between HLA-B27 and ERAP1 Loss-of-function ERAP1 variants confer reduced risk of developing AS in HLA-B27 positive individuals, suggesting an important gene-gene (epistatic) interaction. To investigate the mechanism(s) underlying this effect, we produced a functional ERAP1 knockout in rats using genome editing. In a collaborative project, we have shown that ERAP1 deficiency increases presentation of longer peptides by HLA-B27. Additional preliminary results suggest that ERAP1 deficiency also reduces the formation of disulfide-linked dimers and oligomers (e.g. misfolding) in the ER and attenuates activation of the unfolded protein response. In contrast, HLA-B7 folding is impaired by loss of ERAP1, although this does not lead to misfolding of this allele. Preliminary results also suggest that ERAP1 deficiency confers partial protection from experimental spondyloarthritis in HLA-B27 transgenic rats, mitigating the development of arthritis but not GI inflammation, thus providing a model system to explore effects of ERAP1 loss-of-function of disease. Studies aimed at determining how ERAP1 loss-of-function is protective are ongoing. Osteoblast Mineralization in Axial Spondyloarthritis We generated a number of pluripotent stem cell lines (iPSCs) by reprogramming skin fibroblasts obtained from axial spondyloarthritis patients and healthy controls. IPSCs are pluripotent, and for the purposes of our studies were differentiated into mesenchymal stem cells and then osteoblasts to study whether there are intrinsic differences that may contribute to excess bone formation in this disease. In preliminary studies we have found that patient-derived cells exhibit increased mineralization relative to cells derived from healthy controls. Current efforts are aimed at determining the mechanism(s) responsible for these differences.